The overall goal of this proposal is to engineer (R21) and evaluate (R33) the next generation of a novel probiotic designed to prevent or treat infection by the diarrheagenic pathogen Clostridium difficile and, thereby, limit reliance on antibiotic therapy. Three Specific Aims are proposed for the R21 phase. In Aim 1, we will genetically engineer complete bio-containment into our existing first-generation probiotic, so that it will be lost from the gastrointestinal tract over 3-5 days unless continually dosed. Further, its anti-C. difficile infection (CDI) property will be expressed only upon concomitant sugar-substitute supplementation. In Aim 2, we will assess in vitro properties of the second-generation probiotic. In Aim 3, we will determine colonization efficacy, and also perform proof-of-concept anti-CDI efficacy studies. Quantifiable transition milestones will be available in order to progress to the R33 phase, where two Specific Aims are proposed. In Aim 4, we will determine dosing, delivery and biological containment efficacy. In Aim 5, we will comprehensively evaluate ability of the biotherapeutic agent to prevent or treat CDI (including primary infection and relapse), elicit adaptive immune response(s) to specific antigens, and impact endogenous microbiota recovery following antibiotic suppression or CDI-specific dysbiosis. Thus, the immediate impact of this work will be to verify pre-clinical readiness of our agent, via comprehensive interrogation of its disease-preventing modalities.